MutS1 is a key protein involved in mismatch repair system for ensuring fidelity of replication and recombination in Deinococcus radiodurans. The zwf gene encodes glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate (PP) pathway, which provides adequate metabolites as precursors of DNA repair. In this study, mutS1 and zwf were disrupted by homologous recombination. The zwf mutant (Delta zwf) and the zwf/mutS1 double mutant (Delta zwf/mutS1) were sensitive to ultraviolet (UV) light, H2O2, and DNA cross-linking agent mitomycin C (MMC), whereas the mutS1 mutant (Delta mutS1) showed resistance to UV light, H2O2 and MMC as the wild-type strain. Inactivation of mutS1 resulted in a 3.3-fold increase in frequency of spontaneous rifampicin-resistant mutagenesis and a 4.9-fold increment in integration efficiency of a donor point-mutation marker during bacterial transformation. Although inactivation of zwf had no obvious effect compared with the wild-type strain, dual disruption of zwf and mutS1 resulted in a 4.7-fold increase in mutation frequency and a 7.4-fold increase in integration efficiency. These results suggest that inactivation of the PP pathway decreases the resistance of D. radiodurans cells to DNA damaging agents and increases mutation frequency and integration efficiency in the mutS1 mutant background.